Sensitivity of 2-arachidonoyl glycerol induced vasorelaxation to inhibitors of endocannabinoid hydrolases and cyclooxygenases

W.-S. Vanessa Ho & Michael D. Randall, School of Biomedical Sciences, University of Nottingham, Queen’s Medical Centre, Nottingham NG7 2UH

There have been conflicting reports regarding the action of 2-arachidonoyl glycerol (2-AG) as a vasodilator, perhaps due to its greater susceptibility to degradation than the other major endocannabinoid anandamide. Evidence suggests that 2-AG is primarily hydrolysed by monoacylglycerol lipase (MGL) to arachidonic acid and glycerol, but it might also be metabolized by fatty acid amide hydrolase (FAAH), which is the primary hydrolase for anandamide, and cyclooxygenase (COX). Here , we have examined the effects of inhibitors of FAAH, MGL and COX on the vasorelaxation induced by 2-AG.

Male Wistar rats (200-300g) were stunned by a blow to the back of the head and killed by cervical dislocation. The third-order branches of the superior mesenteric artery (2mm long) were mounted in a wire myograph and maintained at 37 oC in gassed ( 95% O2/5% CO2) Krebs-Henseleit solution (O’Sullivan et al., 2004). Vessels were precontracted with methoxamine (10-30µM). Relaxations to cumulative addition of 2-AG to precontracted vessels are expressed as mean±s.e.m (n≥4 rats) and analysed by two-way analysis of variance of the whole data set. Inhibitors were incubated with vessels for 30 or 45min before determination of a concentration-relaxation curve.

In endothelium-intact vessels, 2-AG induced concentration-dependent relaxation. The relaxations were not affected by the selective FAAH inhibitor, URB597 ( 3’-carbamoyl biphenyl-3-yl-cyclohexylcarbamate ) (control: pEC50=5.4 ± 0.1 E max=89 ±4% ; + 1µM URB597: pEC50=5.5 ± 0.1 Emax=98 ±1% ). In contrast, methylarachidonyl fluorophosphate (MAFP; an inhibitor of FAAH and MGL) enhanced 2-AG relaxations (control: pEC50=5.6 ± 0.1 E max=85 ± 8 %; + 10 µM MAFP: pEC50=5.9 ± 0.2 Emax=100 ±1% ; P<0.001). A similar potentiation effect was also found with the COX-1 inhibitor indomethacin (10µM; pEC50=5.9 ± 0 .1 Emax=86 ± 5%; P<0.05) but not the COX-2 inhibitor nimesulide (10 µM; pEC50=5.6 ± 0.3 Emax=81 ± 10%). Interestingly, the combined treatment of indomethacin and MAFP greatly enhanced 2-AG relaxations, with an apparent additive effect of the two inhibitors (control: pEC50=5.4 ± 0.1 Emax=77 ±

1 5%; + inhibitors, pEC50=6.3 ± 0.3 Emax=100 ±1% ; P<0.001). In endothelium-denuded vessels, the combined treatment also significantly potentiated 2-AG relaxations (control: pEC50=5.3 ± 0.1 Emax=63 ±1 5%; + inhibitors, pEC50=6.4 ± 0.1 Emax=92 ±4% ; P<0.001), but indomethacin alone had no effect (pEC50=5.3 ± 0.1 Emax=62 ±1 3 % ). In contrast to the case for 2-AG, its non-hydrolysable analogue noladin ether caused relaxations that were insensitive to either indomethacin or MAFP (data not shown).

To conclude, in rat isolated small mesenteric artery, relaxations to 2-AG are limited by its degradation via the activity of COX-1 and potentially MGL, but not FAAH. This indicates that cardiovascular actions of 2-AG is likely regulated by local metabolism in the vascular wall.

O’Sullivan SE et al. (2004). Br J Pharmacol141: 803-812.

 This study was supported by the British Heart Foundation (PG/06/064/20985). WSVH is an Anne McLaren Fellow of University of Nottingham.